Superoxide dismutase 1 (SOD1) Mutations cause approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease and transgenic mice expressing mutant SOD1 develop a progressive motor neuron disease. It has been hypothesized that mutant SOD1 damage motor neurons by acquiring a toxic property by the following mechanism: the improperly folded mutant SOD1 catalyze, via peroxynitrites, the nitration of tyrosines; and mutant SOD1 possesses an enhanced peroxidase activity that generates elevated levels of hydroxyl radicals from hydrogen peroxide. In both hypotheses, copper (Cu) bound to mutant SOD1 is proposed to play a critical role, but for a variety of reasons it has been difficult to test the Cu hypothesis. An emerging view is that the delivery of Cu to specific proteins within various intracellular compartments is mediated through distinct Cu chaperones. Recently, it was shown that the delivery of Cu to SOD1 is mediated through a soluble yeast protein termed Lys7 and Cu chaperone for SOD1 (CCS in mammals) First, we will define the cellular distribution s and the axonal transport of Ccs in the nervous system, particularly in motor neurons. Second, to prove that CCS is necessary to charge SOD` with Cu, we will use gene-targeting to delete the gene encoding CCS and characterize the phenotype of CCS null mice. Third, since CCS colocalizes with SOD1, we will determine whether Ccs interacts physically with SOD1 using coimmunoprecipitation studies. Fourth, because CCS levels vary with abundance of SOD1, we will determine the mechanism whereby Ccs is coordinately regulated by SOD1 using a variety of cell culture systems. Our goal is to test whether the aberrant SOD1 Cu chemistry is directly responsible for mediating disease in mutant SOD1 transgenic mice. With information from each of these studies and the availability of CCS null mice, we will be in a unique position to examine directly this hypothesis. If Cu is the key effector of the disease, we predict that CCS null mice that express the FALS-linked mutant SOD1 will show significant amelioration of motor neuron disease. These studies will allow, the examination of Cu-based toxicity mechanisms in vivo; the results of this work, which have the potential to identify new therapeutic targets (i.e., CCS/SOD1 Cu trafficking pathway), will have important implications for the design of treatments for motor neuron disease.